Date of Award

1994

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Eric C. Achberger

Abstract

Intrinsically curved DNA upstream of the RNA polymerase binding site, the promoter, influences transcription in Bacillus subtilis. In this study, curved DNA upstream of the $-$35 region was examined for the ability to influence transcription in Escherichia coli and B. subtilis through the use of hybrid promoters. These promoters were constructed by substituting the curved DNA from two B. subtilis promoters, Bal129 and Alu156, for the DNA upstream of the $-$35 region in the lambda phage promoters $p\sb{\rm R}$ and $p\sb{\rm L}$. The hybrid promoters were designated Bal$p\sb{\rm R}$, Alu$p\sb{\rm R}$, Bal$p\sb{\rm L}$, and Alu$p\sb{\rm L}$. Using transcriptional fusions between each promoter and the chloramphenicol acetyltransferase (cat) gene, CAT expression was measured in B. subtilis and E. coli. The addition of curved DNA significantly increased transcription from the Bal$p\sb{\rm L}$ and Alu$p\sb{\rm L}$ promoters compared with $\lambda p\sb{\rm L}$ in B. subtilis. In E. coli, transcription was not significantly affected by the presence of the curved DNA. In the competition binding assay, E. coli RNA polymerase bound DNA fragments containing the Bal$p\sb{\rm R}$ or Alu$p\sb{\rm R}$ promoters much more effectively than those with the original $\lambda p\sb{\rm R}$. However, the E. coli enzyme did not discriminate among the promoters in the $p\sb{\rm L}$ series. In general, B. subtilis RNA polymerase displayed greater affinity for the hybrid promoters than it did for the original $\lambda$ promoters. DNA supercoiling affected the binding of E. coli RNA polymerase to the hybrid promoters when the interaction was analyzed in a gel retardation assay. On a supercoiled DNA template, the hybrid promoters were bound by RNA polymerase more effectively than was the original $\lambda$ promoters. DNaseI footprinting was used to characterize the interaction between E. coli RNA polymerase and the hybrid promoters. This assay provided evidence that the E. coli RNA polymerase tightly wraps the curved DNA when binding the Bal$p\sb{\rm R}$ and Alu$p\sb{\rm R}$ promoters. No evidence of DNA wrapping was observed with the original $\lambda p\sb{\rm R}$. We propose that the tight wrapping of the upstream curved DNA around the RNA polymerase may facilitate and/or be required for the subsequent steps of DNA untwisting and strand separation.

Pages

115

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